Pelvic inflammatory disease (PID), the frequent infection and inflammation of the female upper genital tract, often results in long-term sequelae, including infertility, chronic pelvic pain, ectopic pregnancy, and recurrent PID. Although Chlamydia trachomatis and Neisseria gonorrhoeae are commonly associated with PID, the etiology of up to 70% of cases is unknown. Emerging cross-sectional evidence implicates Mycoplasma genitalium as a significant etiologic agent of PID, although lack of prospective data makes it impossible to determine if this association is causal. Less in known about the role of other mycoplasmal bacteria, including the newly differentiated Ureaplamsa urealyticum biovar 2 and Ureaplamsa parvum in PID. Similarly, anaerobic bacteria and bacterial vaginosis (BV) have also been associated with PID, yet little is known about the role of several newly identified BV-associated bacteria identified using broad-range PCR and sequencing of bacterial 16S rDNA in PID. As little is known about the role of BV-associated and mycoplasmal bacteria in PID, we propose to test the hypothesis that BV-associated bacteria (Bacterial Vaginosis Associated Bacteria 1 (BVAB1), BVAB2, BVAB3, Gardnerella vaginalis, Atopobium vaginae, Eggerthella-like bacterium, Leptotrichia spp., Megasphaera phylotype 1, Megasphaera phylotype 2, BVAB-TM7, Mobiluncus curtisii, M. mulieris, Peptoniphilus sp. type 1 Peptoniphilus lacrimalis, and Prevotella G1), M. genitalium, and the recently reclassified Ureaplasma biovars are prospectively associated with incident PID. Targeted PCR will be applied to already collected and archived vaginal and endometrial specimens obtained from 141 patients with PID and 423 controls, nested within the prospective Gynecologic Infections and Follow-Through (GIFT) study which followed 1199 high risk adolescents and women three years for the development of PID. The aims of our study are: to determine whether the presence and quantity of BV-associated bacteria and mycoplasmal bacteria in the vagina is predictive of incident PID;to determine the agreement between the presence of BV-associated bacteria and mycoplasmal bacteria identified by PCR in vaginal specimens and endometrial specimens subsequently obtained at the time of PID diagnosis among cases;and to characterize and compare the endometrial microbiota of 44 patients with histologically confirmed endometritis, 57 patients without endometritis, and an external control group of 60 patients undergoing gynecologic surgery for conditions other than PID using broad range 16S rRNA gene PCR. This study, to our knowledge, will be the first to compare the relationships between a broad range of BVassociated and mycoplasmal bacteria, some recently identified and uncultivable, and incident PID. Further, this will be the first broad spectrum PCR investigation using genital specimens collected from patients with PID, in order to potentially identify new bacteria associated with the syndrome. Our study is important, as PID affects more than 1 million women per year and accounts for $9 billion in annual direct and indirect costs. Unfortunately, testing for these bacteria is not routine in clinical practice and some have known resistance to a number of PID treatment regimens, highlighting the importance of this study to raise awareness for the potential need to revise clinical guidelines in order to preserve fertility and prevent chronic pain in patients at risk.